Presentation is loading. Please wait.

Presentation is loading. Please wait.

Figure 5 ISOX and vorinostat partially restore splicing pattern in DM1 patient-derived fibroblasts. (A) ISOX and vorinostat partially rescue mis-splicing.

Published byPrudence Fox Modified over 6 years ago

Similar presentations

Presentation on theme: "Figure 5 ISOX and vorinostat partially restore splicing pattern in DM1 patient-derived fibroblasts. (A) ISOX and vorinostat partially rescue mis-splicing."— Presentation transcript:

1 Figure 5 ISOX and vorinostat partially restore splicing pattern in DM1 patient-derived fibroblasts. (A) ISOX and vorinostat partially rescue mis-splicing of SERCA and INSR in patient-derived DM1 fibroblasts. Human normal fibroblasts (N) and DM1 fibroblasts (DM1) were treated with DMSO (0.1%), ISOX (5 µM) and vorinostat (5 µM) for 2 days. SERCA exon 22 and INSR exon 11 splicing were analyzed by RT-PCR followed by gel electrophoresis. The percentage of exon inclusion is shown below each RT-PCR product. Biological triplicates/replicates of each treatment are indicated by 1, 2 and 3. (B, C) ImageJ quantification of the percent SERCA exon 22 inclusion (B) and INSR exon 11 inclusion (C) from three biological repeats of DM1 fibroblasts and two biological replicates of normal fibroblasts. Error bars show the standard deviation of the biological triplicates/replicates and statistical differences were characterized by a t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. From: A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I Hum Mol Genet. 2017;26(16): doi: /hmg/ddx190 Hum Mol Genet | © The Author Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

2 Figure 4 Compound ISOX and vorinostat increase MBNL1 expression in DM1-patient-derived fibroblasts. (A) Immunoblot images of MBNL1 levels in DM1 patient-derived (top) and normal (bottom) fibroblasts after 5 μM ISOX or 5 μM vorinostat treatment for 2 days. Three biological replicates were generated for each treatment. Tubulin was used as a loading control. (B) MBNL1 levels from A were normalized to tubulin signal and plotted as a bar graph. Error bars show the standard deviation (SD) of the biological triplicates and statistic differences were characterized by a t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. From: A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I Hum Mol Genet. 2017;26(16): doi: /hmg/ddx190 Hum Mol Genet | © The Author Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

3 Figure 3 Cell-based screen identifies small molecules that up-regulate MBNL1 expression. (A, B) Dose response in ZsGreen-MBNL1 cells following ISOX (A) and vorinostat (B) treatment. ZsGreen-MBNL1 and parental HeLa cells were treated with compound and the mean fluorescent intensity (MFI) of the cells was quantified by flow cytometry 48 h after treatment. MFI<sub>HeLa</sub> was subtracted from MFI<sub>ZsGreen-MBNL1</sub> and the resulting MFI relative to DMSO treatment is plotted. (C) Box plot of the ZsGreen-MBNL1 signal changes in CGL screen. Each compound was screened at 1 µM in replicates. The percentage increase of ZsGreen-MBNL1 after compound treatment was calculated as follows: (MFI<sub>compound</sub> -MFI<sub>DMSO</sub>)/(MFI<sub>ISOX</sub> -MFI<sub>DMSO</sub>)×100% and plotted on Y axis. The CGL compounds are divided into gene families (listed on X axis) based on each compound’s primary annotated target activity (at 500 nM or less). The black line in each box represents the mean MBNL1 percentage change; each box indicates the distribution of 50% of compounds around the mean; the whiskers indicate the next 25%, and the outliers are plotted as dots. (D) The percentage increase of ZsGreen-MBNL1 signal after the treatment of top 128 compounds. ZsGreen-MBNL1 and parental HeLa cells were treated with each compound at 1 μM followed by flow cytometry quantifications. The MFI increases were calculated by subtracting MFI<sub>Hela</sub> from MFI<sub>ZsGreen</sub>, and then the percentage increases were calculated and plotted to its primary targeting gene family. Each dot represents one compound. Negative control (0.1% DMSO) did not change MBNL levels (indicated as 0% baseline) and the positive control (5 μM ISOX) increased MBNL1 by 100% (indicated by the red line) on the plot C and D. From: A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I Hum Mol Genet. 2017;26(16): doi: /hmg/ddx190 Hum Mol Genet | © The Author Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

4 Figure 2 Reconstitution of the DM1 phenotype in ZsGreen-MBNL1 cells
Figure 2 Reconstitution of the DM1 phenotype in ZsGreen-MBNL1 cells. Representative fluorescent images of ZsGreen-MBNL1 cells transfected with pcDNA-DMPK-(CTG)<sub>400</sub> plasmid or no construct. (A) Localization of MBNL1 with labeled antibody (red) and ZsGreen-MBNL1 (green) at nuclear foci in (CUG)<sub>400</sub> expressing cells. Scale bar is 2 µm. (B) Fluorescent in situ hybridization with probes against (CUG)<sub>exp</sub> repeats (red) show ZsGreen-MBNL1 (green) is partially co-localized with CAG-Cy3 labeled RNA foci. Nucleus was stained by DAPI (blue). Scale bar is 2 µm. (C) RT-PCR analysis to detect SERCA1 exon 22 alternative splicing in ZsGreen-MBNL1 cells transfected with no plasmid or plasmid expressing (CUG)<sub>400</sub> RNA . Exon 22+ and exon 22− transcripts are shown in the diagram on the right with RT-PCR primer sets indicated by arrows. The band intensity was quantified by ImageJ and the percentage numbers of exon 22 inclusions are listed below. Biological triplicates of each treatment are indicated by 1, 2 and 3. From: A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I Hum Mol Genet. 2017;26(16): doi: /hmg/ddx190 Hum Mol Genet | © The Author Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

5 Figure 1 Site-specific integration of ZsGreen into endogenous MBNL1 locus generates ZsGreen-MBNL1 cells expressing green fluorescent fusion protein. (A) Schematic diagram of the strategy to insert a ZsGreen cassette into the MBNL1 locus (not to scale). The asterisks indicate the position of the single-strand breaks generated by Cas9 nickase/sgRNAs. The middle diagram shows the donor vector that contains the left and right homologous MBNL1 arms and the ZsGreen reporter. (B) ZsGreen integration in MBNL1 locus is confirmed by PCR followed by agarose gel analysis. Primer sets and PCR products are indicated in the upper diagram. (C) Droplet digital PCR (ddPCR) quantifying MBNL1 and ZsGreen copy number in no-template control (NTC), parental HeLa and ZsGreen-MBNL1 genomic DNA and plotted on the bar graph. (D) Immunoblotting shows MBNL1 and ZsGreen-MBNL1 protein expression in parental HeLa and ZsGreen-MBNL1 cells. MBNL1-silencing siRNA (siMBNL1) and non-silencing siRNA (siNS) control were used to demonstrate MBNL1 antibody specificity. Tubulin was used as a loading control. (E) Fluorescent microscopy images of parental HeLa and clonal ZsGreen-MBNL1 #27 using the same exposure. The green fluorescent images are on the left; the merged images of green and blue (DAPI staining) signal are on the right. Scale bar is 20 µm. (F) Flow cytometry quantification of green fluorescent signal in parental HeLa cells and ZsGreen-MBNL1 clone #27. From: A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I Hum Mol Genet. 2017;26(16): doi: /hmg/ddx190 Hum Mol Genet | © The Author Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

Download ppt "Figure 5 ISOX and vorinostat partially restore splicing pattern in DM1 patient-derived fibroblasts. (A) ISOX and vorinostat partially rescue mis-splicing."

Similar presentations

Figure 3. (A) Effect of the ON length on the HTT mRNA knockdown efficiency. LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM concentration into.

Figure 3. (A) Effect of the ON length on the HTT mRNA knockdown efficiency. LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM concentration into.
Figure 1. Schematic of the corA leader mRNA

Figure 1. Schematic of the corA leader mRNA
Figure 1. Single-molecule analysis through microfluidic enrichment of rolling circle amplification products. (A) Sample preparation and target detection.

Figure 1. Single-molecule analysis through microfluidic enrichment of rolling circle amplification products. (A) Sample preparation and target detection.
Figure 2. BRCA2–RAD51 complexes organize into filament-like structures

Figure 2. BRCA2–RAD51 complexes organize into filament-like structures
Table 1 Patient characteristics of attendees of the influenza vaccination, irrespective of AF status From: Yield of screening for atrial fibrillation in.

Table 1 Patient characteristics of attendees of the influenza vaccination, irrespective of AF status From: Yield of screening for atrial fibrillation in.
Fig. 1. Significant clusters in the amygdala and dlPFC, four-way interaction effect (group by valence by temperature by time), small volume corrected.

Fig. 1. Significant clusters in the amygdala and dlPFC, four-way interaction effect (group by valence by temperature by time), small volume corrected.
Figure 1. ChIL-7 gene amplification and its eukaryotic expression vector construction. (A) Chicken IL-7 cDNA gene amplified by RT-PCR: Lane 1, IL-7 gene.

Figure 1. ChIL-7 gene amplification and its eukaryotic expression vector construction. (A) Chicken IL-7 cDNA gene amplified by RT-PCR: Lane 1, IL-7 gene.
Figure 1. Gene expression analysis

Figure 1. Gene expression analysis
Figure 1. RISC activity in BYL. ( A ) Quantity of AGO1 protein in BYL

Figure 1. RISC activity in BYL. ( A ) Quantity of AGO1 protein in BYL
Figure 1. Inhibition of GSK3β reduces MiR biogenesis through repression of pri-MiR processing. (A) qRT-PCR analysis of miR-27a, miR-23a, miR-24, miR-141.

Figure 1. Inhibition of GSK3β reduces MiR biogenesis through repression of pri-MiR processing. (A) qRT-PCR analysis of miR-27a, miR-23a, miR-24, miR-141.
Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells

Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells
From: Dasatinib as a treatment for Duchenne muscular dystrophy

From: Dasatinib as a treatment for Duchenne muscular dystrophy
Figure 2. Synthesis and execution of RNA CHA circuit

Figure 2. Synthesis and execution of RNA CHA circuit
Figure 2. The graphic integration of CNAs with altered expression genes in lung AD and SCC. The red lines represent the amplification regions for CNA and.

Figure 2. The graphic integration of CNAs with altered expression genes in lung AD and SCC. The red lines represent the amplification regions for CNA and.
Figure 1. A3B NTD mediates enzyme activity and oligomerization

Figure 1. A3B NTD mediates enzyme activity and oligomerization
Fig. 1 Nodes in a conceptual knowledge graph

Fig. 1 Nodes in a conceptual knowledge graph
Figure 1: Pneumomediastinum and subcutaneous emphysema as indicated by the arrows. From: Pneumomediastinum and subcutaneous emphysema after successful.

Figure 1: Pneumomediastinum and subcutaneous emphysema as indicated by the arrows. From: Pneumomediastinum and subcutaneous emphysema after successful.
Figure 1. Outline of the TADA setup

Figure 1. Outline of the TADA setup
Figure 1. drb7.2 mutant plants display altered accumulation of endoIR-siRNA. Wild-type (Col-0) and drb7.2 mutant plants were subjected to high throughput.

Figure 1. drb7.2 mutant plants display altered accumulation of endoIR-siRNA. Wild-type (Col-0) and drb7.2 mutant plants were subjected to high throughput.
Figure 2. Temperature-entropy diagram and the flow resistances of the power plant model of Figure 1. From: Thermodynamic optimization of a triple-shaft.

Figure 2. Temperature-entropy diagram and the flow resistances of the power plant model of Figure 1. From: Thermodynamic optimization of a triple-shaft.

Similar presentations

Ads by Google